Hemostatic textile

ABSTRACT

The present invention is directed to a hemostatic textile, comprising: a material comprising a combination of glass fibers and one or more secondary fibers selected from the group consisting of silk fibers; ceramic fibers; raw or regenerated bamboo fibers; cotton fibers; rayon fibers; linen fibers; ramie fibers; jute fibers; sisal fibers; flax fibers; soybean fibers; corn fibers; hemp fibers; lyocel fibers; wool; lactide and/or glycolide polymers; lactide/glycolide copolymers; silicate fibers; polyimide fibers; feldspar fibers; zeolite fibers, zeolite-containing fibers, acetate fibers; and combinations thereof; the hemostatic textile capable of activating hemostatic systems in the body when applied to a wound. Additional cofactors such as thrombin and hemostatic agents such as RL platelets, RL blood cells; fibrin, fibrinogen, and combinations thereof may also be incorporated into the textile. The invention is also directed to methods of producing the textile, and methods of using the textile to stop bleeding.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/620,077 filed Sep. 14, 2012, which is a continuation of U.S. patentapplication Ser. No. 13/411,024 filed Mar. 2, 2012, which is acontinuation of U.S. patent application Ser. No. 11/650,670 filed Jan.8, 2007, which claims the benefit of U.S. Provisional Application Ser.No. 60/758,261 filed Jan. 11, 2006, which is incorporated by referenceherein in its entirety.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention is directed to textiles, such as bandages,sutures, or fabrics, and more particularly to hemostatic textiles thatinclude agents that can control bleeding rapidly and can be stored forlong periods of time.

2. Description of the Related Art

Despite considerable progress in understanding pathophysiologicalprocesses involved in surface (topical) hemostasis, there remains aconsiderable unmet need for materials that can be applied to sites ofhemorrhage to staunch bleeding. Traumatic injury is the leading cause ofdeath for individuals under 44 years of age (Bozeman, W. Shock,Hemorrhage (2001)). Approximately half of the 100,000 deaths annually inthe United States per year from traumatic injury, or 50,000 cases, arefrom exsanguinations, (Peng, R., Chang, C., Gilmore, D. & Bongard, F. AmSurg Vol. 64 950-4 (1998)) and about the same number of hemorrhagingpatients survive after massive red blood cell transfusion (Vaslef, S.,Knudsen, N., Neligan, P., and Sebastian, M. J. Trauma-Inj. Inf. Crit.Care Vol. 53 291-296 (2002)). Thus, approximately 100,000 patients arein critical need of hemorrhage control in the US each year. Thesituation is equally critical in combat casualty care; in a recentreview of military casualties (Burlingame, B. DOD's experiences inAfghanistan Advanced Technological Applications for Combat Casualty Care2002 Conference in www.usaccc.org (2002)), the control ofnon-compressible bleeding was identified as the single most importantunmet need in military emergency medicine. The standard of care isfrequently the application of a tourniquet to control “compressible”bleeding and then gauze to control the residual “noncompressible”bleeding. However, continued blood loss through gauze is a majorcontributor to morbidity and mortality.

The prior art is replete with patents directed to various forms ofbandages. For example, U.S. Pat. No. 3,419,006 to King discloses asterile transparent dressing for a wound and made from a hydrophilicpolymeric gel of an insoluble polymer, and U.S. Pat. No. 4,323,061 toUsukura discloses a rigid bandage made from glass fibers and non-glassfibers. In addition, various methods have been attempted to quicklyarrest bleeding in an injured person. Several of these methods includearticles such as bandages supplemented with substances that chemicallyaccelerate the body's natural clotting processes. Examples of sucharticles include the following:

U.S. Pat. No. 3,328,259 to Anderson discloses a bandage or wounddressing that incorporates polymers such as sodium carboxymethylcellulose, hydroxyethyl cellulose, polyoxyethylene,polyvinylpyrrolidone, and the like.

U.S. Pat. No. 4,192,299 to Sabatano discloses a bandage that includes apacket containing an antiseptic substance.

U.S. Pat. No. 4,390,519 to Sawyer discloses a bandage in the form of asponge and containing collagen or a collagen-like substance.

U.S. Pat. No. 4,453,939 to Zimmerman et al. discloses a compositionuseful as a wound dressing and made from a combination of collagen,fibrinogen and thrombin.

U.S. Pat. No. 4,606,337 to Zimmerman et al. discloses a resorptive sheetfor closing and treating wounds, and composed of a glycoprotein matrixthat contains fibrinogen and thrombin.

U.S. Pat. No. 4,616,644 to Saferstein et al. discloses an adhesivebandage that includes high molecular weight polyethylene oxide as ahemostatic agent.

U.S. Pat. No. 5,800,372 to Bell et al. discloses a dressing made from anabsorbent polymer and includes microfibrillar collagen.

U.S. Pat. No. 5,902,608 to Read et al. discloses surgical aids such asbandages, gauzes, sutures, and the like, that contain fixed-dried bloodcells that express platelet-derived growth factors.

U.S. Pat. No. 6,638,296 to Levinson discloses a bandage that includes apad containing glucosamine or a glucosamine derivative.

U.S. Pat. No. 6,762,336 and International Patent Application PublicationNo. WO/99/59647 to MacPhee et al. discloses a multilayer bandage thatincludes a thrombin layer sandwiched between two fibrinogen layers.

U.S. Pat. No. 6,897,348 to Malik discloses an adhesive bandage thatcontains an antimicrobial agent and a hemostatic agent (e.g., chitosan,niacinamide, or ascorbic acid), or a single wound-healing agent thatcontains both antimicrobial and hemostatic activities (e.g., chitosanniacinamide ascorbate salt).

U.S. Pat. No. 6,891,077 to Rothwell et al. discloses fibrinogen bandagesthat include a procoagulant such as propyl gallate, gallic acid, or aderivative thereof. Optional ingredients such as thrombin or anantimicrobial agent may also be included.

International Patent Application Publication No. WO 97/28823 to NewGeneration Medical Corporation discloses a hemostatic bandage thatcontains powdered fibrinogen and thrombin adhered to a fibrous matrixwith a viscous, nonaqueous adhesive such as a viscous polysaccharide,glycol, or petroleum jelly.

Despite considerable progress in understanding pathophysiologicalprocesses involved in hemostasis, tissue remodeling, and resolution atwound sites, there remains a critical unmet need for a material that canbe applied to sites of injury to accelerate these processes. The presentinvention is believed to be an answer to that need.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed a hemostatic textile,comprising: a material comprising a combination of glass fibers and oneor more secondary fibers selected from the group consisting of silkfibers; ceramic fibers; raw or regenerated bamboo fibers; cotton fibers;rayon fibers; linen fibers; ramie fibers; jute fibers; sisal fibers;flax fibers; soybean fibers; corn fibers; hemp fibers; lyocel fibers;wool; lactide and/or glycolide polymers; lactide/glycolide copolymers;silicate fibers; polyimide fibers; feldspar fibers; zeolite fibers,zeolite-containing fibers, acetate fibers; and combinations thereof; thehemostatic textile capable of activating hemostatic systems in the bodywhen applied to a wound.

In another aspect, the present invention is directed to A hemostatictextile, comprising: a material comprising a combination of about 65% byweight glass fibers and about 35% by weight raw or regenerated bamboofibers; the hemostatic textile capable of activating hemostatic systemsin the body when applied to a wound.

In yet another aspect, the present invention is directed to a hemostatictextile, comprising: a material comprising glass fibers and one or moresecondary fibers selected from the group consisting of silk fibers;polyester fibers; nylon fibers; ceramic fibers; raw or regeneratedbamboo fibers; cotton fibers; rayon fibers; linen fibers; ramie fibers;jute fibers; sisal fibers; flax fibers; soybean fibers; corn fibers;hemp fibers; lyocel fibers; wool; lactide and/or glycolide polymers;lactide/glycolide copolymers; silicate fibers; polyamide fibers;feldspar fibers; zeolite fibers, zeolite-containing fibers; acetatefibers; and combinations thereof; and thrombin or a fraction comprisingthrombin; the hemostatic textile capable of activating hemostaticsystems in the body when applied to a wound.

In yet another aspect, the present invention is directed to a hemostatictextile, comprising: a material comprising a combination of about 65% byweight glass fibers and about 35% by weight raw or regenerated bamboofibers; and from about 0.1 to about 5% by weight of thrombin or afraction comprising thrombin based on the total weight of the textile;the hemostatic textile capable of activating hemostatic systems in thebody when applied to a wound.

In yet another aspect, the present invention is directed to a hemostatictextile, comprising: a material comprising glass fibers and one or moresecondary fibers selected from the group consisting of silk fibers;polyester fibers; nylon fibers; ceramic fibers; raw or regeneratedbamboo fibers; cotton fibers; rayon fibers; linen fibers; ramie fibers;jute fibers; sisal fibers; flax fibers; soybean fibers; corn fibers;hemp fibers; lyocel fibers; wool; lactide and/or glycolide polymers;lactide/glycolide copolymers; silicate fibers; polyamide fibers;feldspar fibers; zeolite fibers, zeolite-containing fibers; acetatefibers; and combinations thereof; and one or more hemostatic agentsselected from the group consisting of RL platelets, RL blood cells;fibrin, and fibrinogen; the hemostatic textile capable of activatinghemostatic systems in the body when applied to a wound.

In yet another aspect, the present invention is directed to a hemostatictextile, comprising: a material comprising a combination of about 65% byweight glass fibers and about 35% by weight raw or regenerated bamboofibers; and one or more hemostatic agents selected from the groupconsisting of RL platelets, RL blood cells, fibrin, and fibrinogen,wherein the RL platelets and the RL blood cells comprise from about 0.1to about 20 wt % and the fibrin and the fibrinogen comprise from about0.1 to about 5 wt %, based on the total weight of the textile; thehemostatic textile capable of activating hemostatic systems in the bodywhen applied to a wound.

In yet another aspect, the present invention is directed to a hemostatictextile, comprising: a material comprising glass fibers and one or moresecondary fibers selected from the group consisting of silk fibers;polyester fibers; nylon fibers; ceramic fibers; raw or regeneratedbamboo fibers; cotton fibers; rayon fibers; linen fibers; ramie fibers;jute fibers; sisal fibers; flax fibers; soybean fibers; corn fibers;hemp fibers; lyocel fibers; wool; lactide and/or glycolide polymers;lactide/glycolide copolymers; silicate fibers; polyimide fibers;feldspar fibers; zeolite fibers, zeolite-containing fibers; acetatefibers; and combinations thereof; and thrombin or a fraction containingthrombin; and one or more hemostatic agents selected from the groupconsisting of RL platelets, RL blood cells; fibrin, and fibrinogen; thehemostatic textile capable of activating hemostatic systems in the bodywhen applied to a wound.

In yet another aspect, the present invention is directed to a hemostatictextile, comprising: a material comprising a combination of about 65% byweight glass fibers and about 35% by weight raw or regenerated bamboofibers; from about 0.1 to about 5% by weight of thrombin or a fractioncomprising thrombin based on the total weight of the textile; and one ormore hemostatic agents selected from the group consisting of RLplatelets, RL blood cells, fibrin, and fibrinogen, wherein the RLplatelets and the RL blood cells comprise from about 0.1 to about 20 wt% and the fibrin and the fibrinogen comprise from about 0.1 to about 5wt %, based on the total weight of the textile; the hemostatic textilecapable of activating hemostatic systems in the body when applied to awound.

In yet another aspect, the present invention is directed to a method ofpreparing a hemostatic textile, comprising the steps of: (1) contactinga material comprising glass fibers and one or more secondary fibersselected from the group consisting of silk fibers; polyester fibers;nylon fibers; ceramic fibers; raw or regenerated bamboo fibers; cottonfibers; rayon fibers; linen fibers; ramie fibers; jute fibers; sisalfibers; flax fibers; soybean fibers; corn fibers; hemp fibers; lyocelfibers; wool; lactide and/or glycolide polymers; lactide/glycolidecopolymers; silicate fibers; polyamide fibers; feldspar fibers; zeolitefibers, zeolite-containing fibers; acetate fibers; and combinationsthereof with thrombin or a fraction comprising thrombin and optionally ahemostatic agent selected from the group consisting of RL platelets, RLblood cells; fibrin, fibrinogen, and combinations thereof; to form a wetmatrix; and (2) drying the wet matrix to produce the hemostatic textile.

In yet another embodiment, the present invention is directed to a methodof preparing a hemostatic textile, comprising the steps of: (1)contacting a material comprising glass fibers and one or more secondaryfibers selected from the group consisting of silk fibers; polyesterfibers; nylon fibers; ceramic fibers; raw or regenerated bamboo fibers;cotton fibers; rayon fibers; linen fibers; ramie fibers; jute fibers;sisal fibers; flax fibers; soybean fibers; corn fibers; hemp fibers;lyocel fibers; wool; lactide and/or glycolide polymers;lactide/glycolide copolymers; silicate fibers; polyamide fibers;feldspar fibers; zeolite fibers, zeolite-containing fibers; acetatefibers; and combinations thereof with platelet rich plasma containingplatelets; (2) crosslinking the platelets; and (3) optionally contactingthe textile with thrombin or a fraction comprising thrombin to producethe hemostatic textile.

These and other aspects will become apparent upon reading the followingdetailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURES

The invention will be better understood when taken in conjunction withthe following figures in which:

FIG. 1 shows representative thrombin generation c s using one embodimentof the present invention;

FIG. 2 shows times for thrombin generation using embodiments of thepresent invention;

FIG. 3 shows thromboelastographic analysis of materials according to thepresent invention;

FIG. 4 shows a comparison of blood cells on dual fiber and gauze;

FIG. 5 shows the interaction of red blood cells (RBCs) with materialsaccording to the present invention;

FIG. 6 shows platelet activation on glass filaments used in the presentinvention; and

FIG. 7 shows total blood loss using the materials of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

The inventors of the present invention have unexpectedly found that ahemostatic textile can be prepared from a composite of glass fibers andone or more secondary fibers. The hemostatic textile made from thecomposite of fibers displays excellent hemostatic properties and fluidabsorbency. To further enhance the hemostatic properties of thehemostatic textile made from the composite, additional blood factorssuch as thrombin, lyophilized blood cells, lyophilized platelets,fibrin, fibrinogen, or combinations of these, may be added. Theseadditional factors aid in activating the body's natural hemostasiscascade and result in a material that can rapidly arrest bleeding. Theinventors have discovered that the combination of glass fibers,secondary fibers, and additional blood factors produce a novelhemostatic textile that rapidly arrests bleeding, and is useful insituations where large hemorrhages exist or when a patient cannot beimmediately admitted to a hospital or trauma treatment center.

The hemostatic textile of the present invention provides importantadvantages over current products that activate hemostasis. The presentinvention is capable of rapidly activating the body's natural hemostaticsystems, such as the blood coagulation cascade, by providing locallyhigh concentrations of substances that activate that cascade. Inaddition, by using lyophilized blood proteins, the hemostatic textile ofthe present invention may be stored in a dry state ready for immediateuse for long periods of time. This aspect is particularly advantageousbecause previous products and systems required hydrated proteins foractivation.

As indicated above, one embodiment of the present invention is ahemostatic textile matrix, comprising a material comprising acombination of glass fibers and one or more secondary fibers. Each ofthese components is discussed in more detail below.

The glass fiber component is preferably a fiberglass prepared byextrusion or electrospinning processes, and has fiber diameters from 5nanometers to 15 microns. Types of glass contemplated for use in thepresent invention include but are not limited to aluminoborosilicateglasses with low sodium oxide content, borosilicate glass, lead glass,aluminosilicate, alkali-barium silicate, vitreous silica, chalcogenideglass, phosphate glass, and bioactive glass sold under the trade name“BIOGLASS”. The dimensions of the glass fiber component may be describedby conventional nomenclature, including the following designations: B(3.5 micron diameter); C (4.5 micron diameter); D (5 micron diameter);DE (6 micron diameter); E (7 micron diameter); G (9 micron diameter); H(10 micron diameter); or K (13 micron diameter). In addition, strandcount of the glass fiber component can range from 900 to 37. The gradeof the glass fiber may be any of electrical grade (“E”), chemical grade(“C”), or high strength (“S”), and the filaments may be in anyarrangement, for example continuous, staple, or textured. The fiberglassfibers may also be used singly or in a plied state using 2 to 20 or morefibers. Fiberglass material is available commercially from varioussuppliers such as Owens Corning, and is available commercially as GradesG75, E-grade fiberglass, and the like, using the designations describedabove.

Secondary fibers used in the textile of the invention include generallyany other fiber that can be combined with the glass fibers to impartabsorbency, softness, and additional hemostatic activity to the textile.As explained in more detail below, use of secondary fibers withexcellent absorbency also aids in incorporating additional hemostaticfactors to the textile. Examples of useful secondary fibers include, butare not limited to, silk fibers; polyester fibers; nylon fibers; ceramicfibers; polysaccharide fibers including plant fibers such as raw orregenerated (e.g., chemically processed) bamboo, cotton, rayon, linen,ramie, jute, sisal, flax, soybean, corn, hemp, and lyocel; animal fiberssuch as wool; lactide and/or glycolide polymers; lactide/glycolidecopolymers; silicate fibers; polyamide fibers; feldspar fibers; zeolitefibers, zeolite-containing fibers; acetate fibers; plant fibers thathave been genetically engineered to express mammalian coagulationproteins or mammalian vasoactive factors. Other secondary fibers thatare suitable for use in the present invention are fibers that have beencovalently modified with polymers to promote water absorbancy (e.g.,polyvinyl alcohols) and polymers that contain molecular moieties thatactivate hemostatic systems (e.g., linear orcyclized-arginine-glycine-aspartate-moieties such as those found ineptifibatide). Preferred secondary fibers include plant fibers such asraw or regenerated (e.g., chemically processed) bamboo fibers, cottonfibers, and the like, that have high moisture absorbancy and that arecapable of activating the intrinsic coagulation cascade. The secondaryfibers may be prepared using conventional methods, including ring, openend (OE), rotor, or air jet spinning, and may have counts ranging from1/1 to 100/1 Ne.

As will be appreciated by one of skill in the art, the secondary fibersmay be used singly, or in combinations of two, three, four, or more in ablended or plied state. In addition, any type of combination ofsecondary fibers may be used. For example, in one embodiment, two ormore secondary fibers may be individually produced and then blended orplied together to form a composite yarn. In another embodiment, thesecondary fibers may be formed as a conjugate comprising blocks of theselected types of fibers, for example alternating blocks of polyestersand polysaccharides. In yet another embodiment, the secondary fibers maybe formed as a homogeneous combination of different threads.

The relative amounts of glass fibers and secondary fibers can rangewidely, for example from about 0.1 to 99.9 wt % glass fibers and about99.9% to 0.1% by weight secondary fibers, based on the total weight ofthe dried textile. Preferable amounts of these materials range fromabout 30 to 80 wt % glass fibers and about 70 to 20 wt % secondaryfibers, and more preferably from about 50 to 80 wt % glass fibers toabout 50 to 20 wt % secondary fibers. Examples of useful proportions ofglass and secondary fibers in the hemostatic textile of the inventioninclude about 50 wt % glass fibers and about 50 wt % secondary fibers;about 40 wt % glass fibers and about 60 wt % secondary fibers; about 30wt % glass fibers and about 70 wt % secondary fibers; or about 20 wt %glass fibers and about 80 wt % secondary fibers. One particularly usefulcombination is about 65% by weight glass fibers and 35% by weight bamboofibers. The glass fiber component and the secondary fiber component maybe combined using conventional methods such as spinning, weaving orknitting, or may be used in a nonwoven state.

In use, the hemostatic textile of the invention may take anyconfiguration. In one embodiment, the hemostatic textile consists of ahemostatic layer designed to accelerate hemostasis, and an outer layerdesigned for surface texture, moisture transfer, fluid adsorption andmicrobial protection. In another embodiment, the hemostatic textileconsists of three layers: a hemostatic layer designed to acceleratehemostasis, a middle layer for bandage strength and elasticity, and anouter layer for designed for surface texture, moisture transfer, fluidadsorption and microbial protection. Additional configurations may beconceived by those of skill in the art.

The hemostatic textile of the invention may also be treated with variousagents that enhance its effectiveness. Examples of additional agentsinclude organic or inorganic compounds that are microstatic ormicrocidal; organic or inorganic compounds that covalently react withblood coagulation proteins; organic or inorganic compounds thatcovalently react with wounded tissue to form covalent bonds for enhancedadhesion to tissues; organic or inorganic compounds that polymerize toform a three-dimensional polymer network at or on the wound; imagingagents such as ultrasound contrast agents (e.g., gas-filledmicrobubbles, metallic nanoparticles, and the like), radio-opaque agents(e.g., iodinated small molecules such as iopromide, iodinated highmolecular weight polymers, and the like), magnetic resonance probes(e.g., ferumoxide iron nanoparticles, superparamagnetic metallicnanoparticles, diethylenetriaminepentaacetate (DTPA)-chelatedgadolinium, and polymers that contain DTPA-chelated gadolinium, and thelike).

Further additional agents that may be included in the hemostatic textileof the invention include skin conditioners such as aloe vera, vitamin E,coenzyme Q, collagen, and the like; anti-inflammatory agents such asaspirin, ibuprofen, acetominophen, vitamin C, COX-2 inhibitors,steroids, and the like; analgesics such as lidocaine, tetrocaine,opiates, cocaine, antihistamines, and the like; antimicrobial orantifungal agents such as bacitracin, silver salts, iodide, and thelike; vasoconstrictors such as epinepherine, norepinephrine,vasopressin, hemoglobin, endothelins, thromboxanes, NO scavengers, andthe like; growth factors such as MMP inhibitors, PDGF, and the like;anti-scar agents such as IL-11, anti-kheloid compounds, and the like;cauterizing agents that undergo an exothermic reaction upon rehydrationsuch as zeolites; dehydrating agents that are hydroscopic such dextran;prothrombotic agents, such as zeolite, dextran sulfate, polyphosphate,mineral interfaces, phosphatidyl serine, calcium, and the like.

The textile matrix of the invention may also include additional factorsthat act to activate the body's natural hemostatic systems and thus aidin quickly arresting bleeding. Such additional factors include thrombinor a plasma fraction that includes thrombin, rehydrated lyophilized (RL)platelets, RL blood cells, fibrin, fibrinogen, and combinations ofthese. In one preferred embodiment, thrombin is incorporated into thetextile to impart additional hemostatic action. The thrombin can be fromany source (naturally isolated, recombinant, etc.) or may be in the formof a plasma fraction or serum that contains thrombin and additionalcoagulation factors such as factor XII, factor XIIa, factor XI, factorXIa, factor XIII, factor XIIIa, factor IX, factor IXa, factor VIII,factor VIIIa, factor vWF, factor V, factor Va, factor X, factor Xa, andcombinations thereof, or other coagulation cofactors such as componentsof animal venom, such as reptilase, or vasoactive agents such asendothelins, thromboxanes, nitrous oxide (NO) scavengers, orcombinations thereof. These factors, or any of the factors listed above,may be in a dry or liquid form when incorporated into the textile of theinvention.

The thrombin contemplated for use in the textile of the invention maytake any form including highly purified thrombin IIa from human oranimal sources, genetically modified plants, or other natural orrecombinant protein expression systems. In addition, partially purifiedthrombin from human or animal sources, genetically modified plants, orother natural or recombinant protein expression systems may be used inthe present invention. The thrombin contemplated for use in the presentinvention may also be contained in purified or partially purified serumor plasma. In one embodiment, the thrombin used in the textile of thepresent invention is a partially purified serum fraction containingthrombin IIa.

The preferred amount of thrombin in the textile of the invention rangesfrom about 0.01% by weight to about 10% by weight, based on the totalweight of the dry textile. More preferred amounts of thrombin includedin the textile of the invention range from about 0.05% by weight toabout 7% by weight, and most preferably from about 0.1% by weight toabout 5% by weight, all based on the total weight of the dry textile.

As explained in more detail in the Examples below, to produce ahemostatic textile that includes thrombin, the textile matrix is soakedin a solution containing thrombin, and frozen and lyophilized.Preservatives such as glycerol, propanediol, polyoxyethylene glycol(PEG) trehalose, and the like, may be included in the soaking solutionto prevent the textile from becoming brittle or chalky duringlyophilization. In general, preservative concentrations in the thrombinsolution range to a maximum of about 20% (v/v). In preferredembodiments, about 12% (v/v) glycerol is used.

In another preferred embodiment, one or more of rehydrated lyophilized(RL) platelets, RL blood cells, fibrin or fibrinogen are incorporatedinto the textile to impart additional hemostatic action. Rehydratedlyophilized blood cells and rehydrated platelets and methods of theirmanufacture are known in the art. See, for example, U.S. Pat. Nos.4,287,087; 5,651,966; 5,891,393; 5,902,608; 5,993,804; all incorporatedby reference herein. Briefly, RL platelets are made by isolating theplatelets, exposing them to a fixative such as formaldehyde, and drying.RL platelets may also be purchased commercially from Entegrion, Inc.(Research Triangle Park, N.C.) under the trade name “STASIX”. Methods ofisolation and purification of fibrin and fibrinogen are also known inthe art.

Briefly, to produce RL blood cells, blood can be obtained from healthyvolunteers, following signed informed consent, incitrate-phosphate-dextrose with adenine (CPDA-1) and subjected tocentrifugation at 1000×g for 20 min to obtain RBCs. The erythrocytes arediluted to a hematicrit=5% in phosphate buffered saline (PBS) andcentrifuged at 2,000×g for 10 min. This step can be repeated twoadditional times to separate RBCs from plasma proteins. RBCs may then becross-linked with glutaraldehyde (for glut-RL RBCs) or a mixture ofparaformaldehyde and glutaraldehyde (for para-RL RBCs). Unreactedaldehyde can be removed from the RBCs by centrifugation (as for theremoval of the cells from plasma proteins), and finally the cells arefrozen and lyophilized at −30° C.

Fibrin and fibrinogen are also available commercially from varioussources. For example, clinical grade material is sold under thetradename HAEMOCOMPLETTAN P from ZLB Behring (Marburg, Germany) andTISSEEL from Baxter (Deerfield, Ill. USA). Research grade material isavailable from Enzyme Research Laboratories (South Bend, Ind. USA).Fibrin and fibrinogen may also be isolated according to procedures knownin the art (e.g., van Ruijven-Vermeer IA, et al., Hoppe Seylers ZPhysiol Chem. 360:633-7 (1979)). Fibrin and fibrinogen may also beisolated using glycine, ammonium sulfate, or ethanol precipitations thatare known in the art.

The RL platelets, RL blood cells, fibrin, or fibrinogen, may be added inpowder form by sprinkling or blowing the dried material onto the matrixand freeze-drying. Alternatively, these materials may be added to thematrix in solution form, and frozen and dried as described above.Preservatives such as glycerol, propanediol, polyoxyethylene glycol(PEG) trehalose, and the like, may be included in the soaking solutionto prevent the textile from becoming brittle or chalky duringlyophilization. In general, preservative concentrations in the thrombinsolution range to a maximum of about 20% (v/v). In preferredembodiments, 12% (v/v) glycerol is used.

Any combination of RL blood cells, RL platelets, fibrin and/orfibrinogen may be incorporated into the textile of the presentinvention. Preferably, the total amount of RL blood cells, RL platelets,fibrin and/or fibrinogen ranges from about 0.1% to about 50% based onthe total weight of the dried textile. In exemplary embodiments, thehemostatic textile of the invention may include the followingcombinations (all weight percents are expressed based on the totalweight of the dried textile):

RL Platelets or RL Blood Fibrin or Cells Fibrinogen Range (wt %) (wt %)Preferred Range 0.1 to 20  0.1 to 5  More Preferred Range 1.0 to 10  0.5to 2  Most Preferred Range 3 to 7 0.75 to 1.5 

In yet another embodiment, the textile matrix of the invention includesboth thrombin or a fraction containing thrombin and one or more ofrehydrated lyophilized (RL) platelets, RL, blood cells, fibrin orfibrinogen. For example, one preferred combination of dried platelets,fibrinogen and thrombin is about 3 to 7 wt % RL platelets, 0.75 to 1.5wt % fibrinogen, and 0.1 to 5 wt % thrombin, all based on the totalweight of the dried textile. In one particularly preferred embodiment, acombination of about 5 wt % RL platelets, about 1 wt % fibrinogen, andabout 0.1 wt % thrombin is used.

A hemostatic textile that contains both thrombin and one or more ofrehydrated lyophilized (RL) platelets, RL blood cells, fibrin orfibrinogen is preferably made by first incorporating thrombin into thematrix followed by incorporation of one or more of rehydratedlyophilized (RL) platelets, RL blood cells, fibrin or fibrinogen usingthe techniques described generally above. In one embodiment, thehemostatic textile of the invention may be infused with a combination offibrinogen and thrombin as disclosed in U.S. Pat. No. 6,113,948,incorporated herein by reference, and available from ProFibrix BV(Leiderdorp, The Netherlands) under the trade name “FIBROCAPS” (acombination of fibrinogen microspheres and thrombin microspheres).Preservatives such as glycerol, propanediol, polyoxyethylene glycol(PEG) trehalose, and the like, may be included in the soaking solutionto prevent the textile from becoming brittle or chalky duringlyophilization. In general, preservative concentrations in the thrombinsolution range to a maximum of about 20% (v/v). In preferredembodiments, 12% (v/v) glycerol is used.

Generally, the hemostatic textile of the invention is made by thefollowing steps:

-   -   1. RL platelets or RL blood cells are prepared and lyophilized        according to published procedures;    -   2. The hemostatic textile is manufactured from textile        components. During this step, the textile may be treated        chemically by addition of a defined amount of agents such as        glycerol, propanediol, polyoxyethylene glycol (PEG) to preserve        the textile and aid in adhesion of hemostatic proteins.        Additionally, in this step, thrombin-containing serum or plasma        is freeze-dried onto the textile matrix.    -   3. Hemostatic proteins such as RL platelets, RL blood cells,        fibrin, or fibrinogen are applied directly to a selected surface        of the hemostatic textile (e.g., a surface that will contact        wounded tissue) at a pre-selected particle density (protein per        square area of textile surface) or weight percentage based on        the total weight of the textile. The hemostatic proteins may be        applied in any order, and may be applied to the textile in        solution form or in a dry form. In one embodiment, RL platelets        may be aldehyde-stabilized, applied to the textile in a liquid        state, and then freeze dried onto the textile.    -   4. The infused hemostatic textile is packaged and optionally        subjected to sterilization (e.g., gamma or UV irradiation).

Detailed examples of hemostatic textiles and their method of manufactureare outlined below.

The textile matrix of the invention is capable of activating hemostaticsystems in the body when applied to a wound, including the bloodcoagulation systems and vasoconstriction systems. It has long been knownthat various materials activate platelets and other blood coagulationfactors when they come into contact with a wound site. Platelets, as aprimary cellular component of blood that provide hemostasis in responseto vascular injury, become contact-activated when exposed to foreignmaterials such as metals glass, and plastics. See, for example, Barr, H.The stickiness of platelets. Lancet ii, 775 (1941)). In addition, it iswell known that thrombin converts fibrinogen to fibrin in the bloodclotting cascade. The combination of components in the hemostatictextile of the present invention act together locally andsynergistically to activate the blood coagulation cascade in a highlyconcentrated and localized form when applied to a wound.

The hemostatic textile of the invention is useful as a wound dressing,for example, a bandage, gauze, and the like, or may be shaped intosutures for use in surgery. Additional uses include forming thehemostatic textile of the invention into fabrics for use in themanufacture of protective clothing or liners for clothing, or for use intourniquets. Additionally, in another embodiment, the hemostatic textileof the present invention is in the form of a kit for use in surgery oremergency or trauma situations. The kit includes the hemostatic textileof the invention in rolls, sheets, or other appropriate shape, and maybe used with or without the additional blood factors.

EXAMPLES

The following examples are intended to illustrate, but in no way limitthe scope of the present invention. All parts and percentages are byweight and all temperatures are in degrees Celsius unless explicitlystated otherwise.

Materials

The following solutions were used in the Examples described below.

Anticoagulant Citrate Dextrose (ACD): 0.042 M Na₃Citrate, 0.035 M citricacid, 20% (w/v) anhydrous dextrose, pH to 4.5.

Citrated Saline: 6.2 mM Na₃Citrate, 150 mM NaCl, pH 6.5.

Imidazole Buffered Saline: 84 mM imidazole, 150 mM NaCl, pH 6.8.

4% Paraformaldehyde: 20 grams paraformaldehyde and 9.4 grams NaH₂PO₄ aresuspended in 400 ml deionized H₂O and heated to about 60° C. in a waterbath until dissolved. pH is set to 7.2 and water is added to 500 ml.

Fixative Solution (prepared immediately before use): Combine 1 ml ACD,10 ml 0.135 Molar NaH₂PO₄, pH 6.5, and 9 ml 4% (w/v) paraformaldehyde.

Imidazole Buffer: 84 mM imidazole, pH=6.8

Citrate Stock: 3.2% sodium citrate, pH=7.4

Examples 1-7: Preparation of Hemostatic Textiles

The following specific textile combinations was made and used in theexperiments that follow:

Textile 1: Woven style; G75 fiberglass in warp; 30/1 100% Bamboo RayonOE in the Fill

The above combination was a glass fiber/bamboo co-weave with the glassfibers (G75, electronic grade E225 spun yarn from E-grade extrudedglass) in the “long” orientation (warp) and the bamboo fibers running“across” the fabric (fill).

Textile 2: Woven Style; ECBC150 1/0 1.0 Z Fiberglass in warp; 18/1 100%Bamboo Rayon MJS in the fill.

Textile 3: Woven Style; ECE225 2/0 4.0 Z Fiberglass in warp; 18/3 100%Bamboo Rayon RS in the fill.

Textile 4: Knit Style; 1 end—ECG75 1/2 Fiberglass; 1 end—18/1 100%Bamboo Rayon OE in the fill.

Textile 5: Knit Style; 2 Ply—ECG150 1/0 Fiberglass twisted with 20/1100% Bamboo Rayon.

Textile 6: Woven Style; ECE225 2/0 4.0 Z Fiberglass in warp; 16/2 100%Flax in the fill.

Textile 7: Woven Style; ECH18 1/0 0.7 Z Fiberglass in warp; 18/2 100%Lyocel MJS in the fill.

Example 8: Preparation of a Hemostatic Textile Matrix that IncludesThrombin

120 mg angel hair grade glass fiber was combined with 8 ml plasma(Innovative Research, Inc., Southfield, Mich.) and 80 μm of 1M CaCl₂ andplaced on rocker. The mixture was mixed gently on the rocker for about90 minutes, and the glass fiber separated from the mixture bycentrifugation (300×g for 5 minutes). The supernatant was collected andglycerol was added to a final concentration of 9% by weight. The finalproduct is a serum that contains thrombin IIa.

50 cm² of Textile 1 above was soaked for about 1 minute in approximately5 ml of the above serum. Excess serum was allowed to drain off, and thesoaked textile matrix was frozen at −20° C. and lyophilized.

Example 9: Preparation of a Hemostatic Textile Matrix that IncludesThrombin and an Additional Hemostatic Agent

A. Textile Matrix that Includes Thrombin and Rehydrated Lyophilized (RL)Platelets

Two methods for preparing textile-based hemostatic matrixes withrehydrated lyophilized (RL) platelets and thrombin are detailed here.The quality of preparations may be evaluated using the ThrombinGeneration Analysis described below.

1. Method 1

In this method, RL platelets are manufactured separately, then added tothe textile matrix.

(a). Preparation of RL Platelets

RL platelets are prepared as described in U.S. Pat. Nos. 5,651,966 and6,139,878, herein incorporated by reference. Alternatively, thefollowing procedure may be used:

Platelet rich plasma (PRP) is prepared by first drawing fresh venousblood into ACD (e.g., 42.5 ml blood into 7.5 ml ACD in a 50 cc syringe).The blood is subjected to centrifugation (10 minutes, 1,200 rpm at25-27° C.) to layer the red blood cells. The PRP is found in thesupernatant. Alternatively, PRP can be isolated from stored and/oroutdated platelets by removing the cells from the storage bag andcentrifuging for 10 minutes at about 1200 rpm to remove contaminatingred blood cells and aggregated platelets.

Plasma proteins are removed from PRP using centrifugation (10 minutes atabout 2400 rpm), and resuspending the isolated platelets in citratedsaline buffer. The platelets are washed twice in citrated saline buffer,and resuspended in citrated saline buffer to a final concentration ofabout 8×10⁹ platelets/ml. Alternatively, plasma proteins may be removedfrom PRP using sizing chromatography (Sepharose 4B equilibrated withcitrated saline buffer). Turbid fractions can be collected, combined,and platelets isolated by centrifugation. The platelets are thenresuspended in citrated saline buffer to a final concentration of about8×10⁹ platelets/ml.

The isolated platelets are cross-linked by adding dropwise 3.5 ml offixative solution to 1.25 ml of isolated platelets at a concentration ofabout 8×10⁹ platelets/ml with gentle stirring. Fixative solution isadded to a final volume of about 10 ml, and the mixture is incubated for1 hour at room temperature without stirring or agitation. Thecross-linked platelets are isolated by centrifugation (10 min at 2400rpm), and resuspended in imidazole buffered saline, washed, and finallyresuspended in imidazole buffered saline to a final concentration ofabout 1×10⁹ platelets/ml.

The crosslinked platelets are frozen and lyophilized by first suspendingthem in imidazole buffered saline with 5% bovine serum albumin at pH 6.8to a final platelet concentration of about 0.8×10⁹ platelets/ml. Themixture is distributed into 1 ml aliquots, frozen at −80° C., andlyophilized overnight or longer. The lyophilized product may be storedat −20° C. The lyophilized cross-linked platelets may be rehydrated byresuspending in imidazole buffer.

(b). Addition of RL Platelets to a Matrix.

To prepare a matrix that contains RL, platelets, RL platelets are firstmilled in the dry state to a fine powder. The fine powder is sprinkledevenly onto a flat sterile surface, such as a Petri dish and one side ofthe freeze-dried thrombin-loaded matrix prepared in Example 8 above ispressed onto the RL powder and removed. The powdered RL platelets mayalso be blown into the textile using known blowing techniques. As analternative method, RL platelets can be prepared without freeze-dryingand in the absence of serum albumin, and resuspended in the thrombinserum prepared in Example 8 above. Soaking the desired matrix in thissolution produces the final product. As a second alternative, rehydratedRL platelets can be resuspended in the thrombin serum described inExample 8 above and used to soak the desired matrix to product the finalproduct.

2. Method 2

In this method, the RL platelets are aldehyde-stabilized after bindingto the textile matrix, as a component of the matrix. The principle hereis to first allow the platelets to contact-activate and adhere to thematrix through normal interactive processes between the hemostatic cellsand the textile fibers. The platelets and textile matrix is then takenthrough the aldehyde stabilization process together. This methodincludes the following steps:

-   -   (a) Platelet rich plasma (PRP) is prepared as described above or        alternatively obtain normally liquid stored platelet rich        plasma.    -   (b) The platelet rich plasma is incubated with a quantity of the        textile matrix that has been predetermined to bind 90% of the        platelets. To predetermine the extent of binding, a sample of        matrix is incubated with excess platelets (more than enough to        saturate the matrix) and the amount of platelets left in the        mixture after the matrix is removed is calculated.    -   (c) The matrix is removed from the PRP and the platelet        concentration in the residual fluid is measured.    -   (d) The textile is incubated in a 10× volume of citrated saline        for 5 min on a rocker and excess fluid is drained. This step is        repeated three times. The soaked textile matrix is then placed        in an appropriate volume of citrated saline for a bound textile        platelet count of 8×10⁹ platelets/ml for use in the crosslinking        step described below.    -   (e) 3.5 ml of fixative solution is dropwise added to 1.25 ml        soaked platelet-matrix (at 8×10⁹ platelets/ml) with gentle        swirling to mix. The mixture is further diluted to a final        volume of 10 ml with a faster addition of fixative solution for        a final bound textile platelet count of 1×10⁹ platelets/ml.        Incubation is performed for 1 hr at room temperature without        stirring or agitation. Finally, the platelet-textile matrix is        diluted into a ten volumes of imidazole buffered saline and        incubated for 5 min on a rocker, and excess fluid is drained        off. This step is repeated three times.    -   (f) To include thrombin, excess imidazole buffered saline is        removed from the platelet-textile matrix as thoroughly as        possible and then soaked in excess IIa serum as described above.        Excess IIa serum is removed and the textile matrix is frozen and        lyophilized as described above.    -   (g) Characterize the freeze-dried hemostatic matrix using        thrombin generation analysis (described below).

It will be appreciated by those of skill in the art that althoughExamples 8 and 9 utilize Textile 1 above to prepare one embodiment ofthe present invention, any textile combination described herein mayalternatively be used (e.g., textiles 2-7 described above).

Thrombin Generation Analysis

This procedure follows Fischer, T. H. et al. Synergistic plateletintegrin signaling and factor XII activation in poly-N-acetylglucosamine fiber-mediated hemostasis. Biomaterials 26, 5433-43 (2005).Briefly, the kinetics of thrombin generation can be used to reflect theability of hemostatic matrixes to function as a catalytic surface forcomponents (e.g., factor XII) of the coagulation cascade. In this assay,thrombin (IIa) cleaves the non-fluorescent synthetic substratepeptide-D-Phe-Pro-Arg-ANSNH to generate a fluorescent product. Thetimecourse for fluorescence generation is followed in a 96-wellfluorescent platelet reader in kinetic mode.

96 well plates are blocked with 150 μl 5% BSA and citrated salineovernight at 37° C., then stored at 4° C. until use. 4 mm (approx.)diameter pieces of hemostatic matrix are prepared with a 4 mm Trephinepunch or razor or sharp scissor. Fluorometric IIa substrateD-Phe-Pro-Arg-ANSNH (Cat #SN-17a-C₆H₁₁ from Haematologic Technologies,Inc., Essex Junction, Vt.) is diluted 1/200 into plasma, followed byCaCl₂ to a final concentration of 10 mM. The mixtures are placed into afluorometer and fluorescence (490 nm) measured for approximately twohours. Data are analyzed by plotting the timecourse from each well, andmeasuring the initial and maximal slope of the relative fluorescencechange curve and the time required to obtain the maximal slope. Theinitial and maximal slopes, together with the time to maximal slope, arethe quality metrics. The higher the slopes and the shorter the time tomaximal slope the more pro-hemostatic the matrix.

Example 10: Preparation of a Dual Fiber Textile

This Example illustrates preparation of a textile made from glass fibersin combination with another selected textile fiber. Two lines ofevidence point towards continuous filament glass thread as being apotential component of a hemostatic textile. First, platelets were foundto activate and adhere to glass (Barr, H. Lancet 238, 609-610 (1941)).As a consequence, glass vessels are generally avoided in the in vitrohandling of platelets, and binding to glass is a longstanding method forassessing platelet activity (McPherson, J. & Zucker, M. B. Blood 47,55-67 (1976); Tsukada, T. & Ogawa, T. Rinsho Ketsueki 14, 777-84 (1973);Cooper, R. G., Cornell, C. N., Muhrer, M. E. & Garb, S. Tex Rep Biol Med27, 955-61 (1969)). Secondly, plasma proteins (Stouffer, J. E. &Lipscomb, H. S. Endocrinology 72, 91-4 (1963); Lissitzky, S., Rogues, M.& Benevent, M. T. C R Seances Soc Biol Fil 154, 396-9 (1960); Bull, H.B. Biochim Biophys Acta 19, 464-71 (1956)), FXII (Ratnoff, O. D. &Rosenblum, J. M. Am J Med 25, 160-8 (1958)) and fibrinogen (Sit, P. S. &Marchant, R. E. Thromb Haemost 82, 1053-60 (1999); Rapoza, R. J. &Horbett, T. A. J Biomed Mater Res 24, 1263-87 (1990); Perez-Luna, V. H.,Horbett, T. A. & Ratner, B. D. J Biomed Mater Res 28, 1111-26 (1994))being well-studied examples, undergo chemical and physical adsorptionprocesses on foreign surfaces (Silberberg, A. J Physical Chem. 66,1872-1883 (1962)). FXII (Hageman Factor) was found to be particularlyimportant because it initiates the humoral coagulation at theglass/blood interface (Ratnoff, supra; Ratnoff, O. D. & Margolius, A.,Jr. Trans Assoc Am Physicians 68, 149-54 (1955)). Platelet activationand turnover of the intrinsic coagulation are highly interrelatedmechanisms, in part due to the role platelets play as a catalyticsurface for assembly of the Va/Xa complex for thrombin generation.Platelet activation by biomaterials (e.g., via integrin outside-insignaling) can result in surface presentation of phosphatidylserine, animportant component of catalytic complexes for thrombin generation(Fischer, T. H., Connolly, R., Thatte, H. S. & Schwaitzberg, S. S.Microsc Res Tech 63, 168-74 (2004)). Factor XII has been found to beperipherally associated with the platelet surface for activation of theintrinsic coagulation pathway in the microenvironment of the cell(Iatridis, P. G., Ferguson, J. H. & Iatridis, S. G. Thromb DiathHaemorrh 11, 355-71 (1964); Shibayama, Y., Reddigari, S. & Kaplan, A. P.Immunopharmacology Vol. 32 24-7 (1996)), although the series ofproteolytic steps involving factor XII that occurs on the plateletsurface are poorly understood. The net effect of the close relationshipbetween XIIa-mediated coagulation and platelet activation is synergismfor earlier initiation of fibrin polymerization.

The interaction of fluids with glasses are largely controlled by surfacetension phenomena related to hydrophobicity, zeta potential andwettability. There is minimal interaction of fluids with the interior ofthe filaments. Thus, a second type of more absorptive fiber was soughtto compensate for the low fluid transport and absorptivity of glass. Apanel of natural and synthetic fibers was tested for tendency toactivate platelets and the intrinsic coagulation cascade. A dual fiberproduct consisting of continuous filament type E-glass and specialtyrayon specialty rayon was prepared and tested for hemostaticeffectiveness in porcine models for hemorrhage.

Materials Type E continuous filament glass (ECDE 11.6 fiberglass) wasprovided by Carolina Narrow Fabrics, Inc. (Winston-Salem, N.C.).Specialty rayon made from bamboo (Bambusa textilis) and other naturaland synthetic fibers were provided by Cheraw Yarn Mills, Inc. (Cheraw,S.C.). The dual fiber glass/specialty rayon textile fabric was preparedby Carolina Narrow Fabrics, Inc. (Winston-Salem, N.C.). Gauze wassourced from Kendall (Mansfield, Mass.).Platelet rich Plasma Isolation Peripheral blood from consenting normalvolunteers was drawn into citrate anti-coagulant, then platelet richplasma was isolated with differential centrifugation as detailed inFischer, T. H. et al. Biomaterials 26, 5433-43 (2005). The plateletconcentration in the platelet rich plasma was measured with a Hiskahaematological analyzer, and the platelet concentration was adjusted to150,000 platelets/ul by diluting the sample with platelet free plasma.Thrombin Generation Kinetics The effect fibers on the kinetics ofthrombin generation in platelet rich plasma (at 150,000 platelets/ul)was investigated by following the hydrolysis of the thrombin substrateD-Phe-Pro-Arg-ANSNH to yield a fluorescent reaction product. 300 ug ofeach fiber was tested in 100 ul platelet rich plasma in triplicate withthe fluorogenic substrate D-Phe-Pro-Arg-ANSNH. The time-course forthrombin generation was initiated by adding CaCl₂ for 10 mM to eachsample. The lag time for thrombin generation was defined as the timepoint at which the fluorescence increased 10% over the initial baselinevalue.Thromboelastography Thromboelastographic (TEG) measurements wereperformed with a TEG-5000 Thrombelastograph Hemostasis Analyzer(Haemoscope Corporation, Niles, Ill.). The assays were initiated byadding CaCl₂ to 10 mM to platelet rich plasma (at 150,000 platelets/ul)and then immediately transferring 327 ul of the calcified platelet richplasma to the thromboelastography chamber that contained the materialsin 33 ul citrated saline. The final fiber concentration was 3.0 mg/ml.Measurements were performed for one hour in triplicate at 37° C., andthen relevant parameters were extracted from the “stiffness” curve.Scanning Electron Microscopy SEM analysis on glucosamine-based materialswas performed as follows. Whole peripheral blood from normal humanvolunteers was allowed to flow directly from the venapuncture butterflyonto the dual-fiber textile or gauze so that 1 cm×1 cm of each materialwas covered by 2 ml of whole blood. The materials were allowed toincubate for one minute, then diluted to 50 ml with citrated saline +1mM EGTA to quench hemostatic processes. The materials were allowed tosettle for 5 min with gravity, then rediluted with citrated saline. Thisprocess was repeated two more times to obtain each material free ofunbound RBCs. After one-minute contact with blood and the multiplecycles of dilution and material/RBC complex settling, glutaraldehyde wasadded for 0.1% (w/v) and the samples were allowed to incubate at roomtemperature for one hour. The samples were diluted 1/1 (v/v) with 4%paraformaldehyde for a final concentration of 2%, and then additionalglutaraldehyde was added for a final concentration of 0.5%. The initialstabilization step with 0.1% glutaraldehyde has been shown to minimizeosmotically driven alterations in red blood cell (RBC) morphological dueto paraformaldehyde exposure (Fischer, T. H. et al. Microsc Res Tech 65,62-71 (2004)). Samples were stored at 4° C. overnight and then examinedwith a Cambridge S200 scanning electron microscope at 20 kv.Measurement of Bound RBCS—10 mg samples of dual fiber or gauze weredirectly exposed to 1.0 ml whole peripheral blood and washed as detailedfor scanning electron microscopy. The pGlcNAc were then placed in 10 mldistilled water with 1% TX-100 to release hemoglobin from bound RBCs.The samples were centrifuged at 10,000×g for five minutes, then theabsorbance at 414 nm was measured to quantify the total amount ofhemoglobin (and thus number of RBCs) associated with each material.Measurement of Extent of RBC Lysis Due to Material Contact 10 mg samplesof dual fiber or gauze were exposed to 1.0 ml whole peripheral blood asin the last two sections. After one minute of exposure, the samples werecentrifuged at 10,000×g for five minutes to pellet blood cells and othermaterials. The optical density at 414 nm was measured to quantify theamount of released hemoglobin in the supernatants, and thus the amountof shed blood.Porcine Brachial Plexis and Femoral Artery Transection Hemorrhage Model40 to 50 kg mixed breed pigs are anesthetized with isofluorane and thenseveral sensors are placed to follow hemodynamic and vasoactiveprocesses: a pulmonary artery thereto dilution catheter is inserted viathe external jugular vein into a pulmonary artery; micromanometer-tippedcatheters are positioned via the left femoral vessels into the rightatrium and thoracic aorta; a 22 gauge catheter is inserted into the leftfemoral artery and connected to a withdrawal pump; catheters arepositioned via the left femoral vessels.

The hemorrhage-challenge phase of the experiment was performed in twophases. First, the transactional laceration of contralateral brachialarteries was performed. The brachial arteries and two associated ˜3 mmdiameter veins were surgically exposed. One each side the artery and twoveins were completely transected with a single scalpel stroke. Injurieswere established in a near simultaneous manner, and then each side wasimmediately packed with the dual fiber textile or gauze. The penetratingcut-down sites were completely packed with each material and thenpressure was held for six minutes. Packing was removed and the amount ofshed blood was ascertained as described below. Both wounds were thenrepacked with the dual fiber material to stabilize the animals. Thesecond phase of the experiment proceeded by surgically exposingcontralateral the femoral arteries. The two contralateral femoralarteries were transected in a near-simultaneous manner and then thesurgical sites were packed with dual fiber textile or gauze. Pressurewas held for six minutes, then materials were removed for shed blooddetermination.

The amount of blood shed into the original packing materials weremeasured by placing the dual fiber or gauze into one liter of distilledwater to lyse RBCs. After two hours of stirring at room temperature andstorage at 2° C. the optical density at 414 nm was measured to determinethe amount of released hemoglobin, and thus the number of shed RBCs andvolume blood loss.

Results

Experiments proceeded in three stages. First, candidate materials forformulating the hemostatic textile were identified by measuring theability of selected fibers to activate hemostatic processes in plateletrich plasma. Secondly, a dual fiber combination was subjected to TEG andSEM analysis to gain insight into the mechanism of function. Finally,the ability of the dual fiber textile to provide hemostasis with porcinehemorrhage models was assessed. Details of these experiments follow.

Activation of Hemostatic System by Candidate Fibers A panel of commontextile fibers were analyzed for their ability to activate platelets andaccelerate turnover of the intrinsic (contact) coagulation pathway. Thebehavior of the representative fibers in the fluorogenic thrombingeneration assay is depicted in FIG. 1. In FIG. 1, duplicate samples ofglass, specialty rayon or gauze fibers were placed in platelet richplasma that contained a fluorogenic thrombin substrate and then thethrombin generation timecourse was initiated by adding calcium. Arrowsindicate the times for thrombin generation. As shown in FIG. 1, exposureof the platelet rich plasma to type E continuous filament glass resultedin thrombin generation in approximately eight minutes. Specialty rayonwas less prothrombogenic with thrombin generation occurring in 12minutes, while gauze fiber was considerably slower.

The behavior of a more expansive panel of fibers is presented in FIG. 2.In FIG. 2, the indicated fibers were tested as detailed in FIG. 1 tomeasure times for thrombin generation. Error bars represent the standarddeviation of duplicate analysis. As shown in FIG. 2, glass and specialtyrayon were respectively the first and second most thrombogenic materialtested. Chitin and gauze, which are components of products for surfacehemostasis, did not strongly accelerate thrombin generation. A prototypebandage was thus constructed from glass and specialty rayon.

In Vitro Properties of the Glass/Specialty Rayon Dual Fiber Textile Thedual-fiber matrix and gauze were compared in the thromboelastographicanalysis with platelet rich plasma depicted in FIG. 3. In FIG. 3, dualfiber or gauze were placed in the thromboelastographic cuvette withnormal saline and then platelet rich plasma and calcium were added toinitiate the clot formation timecourse. Normal saline without materialwas run as a negative control. As shown in FIG. 3, the glass/specialtyrayon textile was found to significantly accelerate fibrin clotformation as compared to gauze or the saline control. Analysis of thedual fiber matrix and gauze after contact with excess peripheral bloodwas performed using scanning electron microscopy as shown in FIG. 4. InFIG. 4, dual fiber or gauze was saturated with excess peripheral bloodand then examined with scanning electron microscopy as detailed above.White arrows in the left two dual fiber panels indicate specialty rayonfibers. As shown in FIG. 4, the glass/specialty rayon matrix tightlybound significant numbers of RBCs, while these cells only sparselycovered the gauze matrix.

Quantification of the number of RBCs on each matrix is shown in FIG. 5.In FIG. 5, dual fiber or gauze was saturated with excess peripheralblood, and then the number of bound RBCs was measured as describedabove. Error bars represent the standard deviation from duplicatemeasurements. As shown in FIG. 5, the dual fiber textile boundapproximately ten times as many RBCs as gauze. Significant lysis of RBCsdid not occur (data not shown). SEM examination of the dual fiber matrixalso showed a large number of highly activated platelets on thecontinuous glass filament component, as shown in FIG. 6, which showsdual fiber saturated and washed with peripheral blood and examined withscanning electron microscopy. These results indicate that theglass/specialty rayon matrix is more effective at providing surfacehemostasis than gauze.

Ability of the Dual Fiber Bandage to Provide Hemostasis in PorcineModels The dual fiber matrix was compared to gauze in severe porcinelarge vessel transection injuries. Two types of injuries wereestablished on each of four pigs. First, the brachial artery and twoassociated large veins in contralateral plexus areas were completelytransected in a near-simultaneous manner. This gives an exsanguinatinghemorrhage that is both arterial and venous in nature. The contralateralcut-down/injury sites were immediately packed with as much dual fibertextile or gauze as required to fill the injury site. Pressure was thenheld for six minutes, and then the sites were unpacked and the degree ofhemostasis was judged as complete (with no visible hemorrhage), partial(with less than three ml of blood loss per minute) or uncontrolled (withmore than three ml of blood loss per minute). The blood loss ontopacking materials and any shed blood was measured from each wound site.The contralateral brachial plexus injury sites were then re-packed withdual fiber textile so as to stabilize the animal for the second set offemoral injuries. Contralateral femoral arteries were exposed, and thencompletely transected in a near simultaneous manner to initiate anexsanguinating hemorrhage. As with the brachial plexus injuries, theinjury sites were immediately packed with dual fiber textile or gauze.After holding pressure for six minutes the sites were unpacked and thedegree of hemostasis was judged as described for the brachial injuries.An important feature of this large vessel transection model is that theanimals were not in hemorrhagic shock. Because the injuries wereimmediately packed with pressure, the mean arterial pressure wasmaintained in the 45 to 55 mm. Hg range and total blood loss was notmore than ˜5% of the total blood volume. The total amount of blood losswith the dual fiber material was approximately half as with gauze withboth brachial plexus and femoral injuries as shown in FIG. 7. In FIG. 7,the total amount of material-absorbed and shed blood from the six minutepressure period was measured from the brachial (Left Panel A) andfemoral (Right Panel B). Error bars represent the standard deviation ofthe blood loss from similar injuries in five animals. As shown in FIG.7, there was a marked tendency for gauze to pull off the hemostatic plug(to the extent that there was one), while the dual fiber textile did notstrongly incorporate into the hemostatic zone.

The above results show that fundamental principles of hemostasis can beused to design economical materials for surface hemostasis. Theglass/specialty rayon textile outperformed gauze in porcine models ofboth capillary and large vessel injury; bleed times and blood loss werereduced by approximately halve when the fiber component of the textilewas optimized with respect to thrombogenicity.

While the invention has been described above with reference to specificembodiments thereof, it is apparent that many changes, modifications,and variations can be made without departing from the inventive conceptdisclosed herein. Accordingly, it is intended to embrace all suchchanges, modifications, and variations that fall within the spirit andbroad scope of the appended claims. All patent applications, patents,and other publications cited herein are incorporated by reference intheir entireties.

What is claimed is:
 1. A packaged hemostatic textile comprising: atextile material comprising glass fibers cowoven or knitted with bamboorayon fibers, wherein when the textile material is in contact with awound on an animal or human, hemostatic systems are activated in theanimal or human, and wherein the textile material is packaged in asterilized package.
 2. The packaged hemostatic textile of claim 1,wherein the glass fibers comprise continuous glass fibers.
 3. Thepackaged hemostatic textile of claim 1, wherein the glass fibers have adiameter of between 5 nanometers and 15 microns.
 4. The packagedhemostatic textile of claim 1, wherein the textile material comprisesthrombin or a fraction containing thrombin.
 5. The packaged hemostatictextile of claim 1, wherein the textile material comprises a hemostaticagent selected from the group consisting of rehydrated lyophilizedplatelets, rehydrated lyophilized blood cells, fibrin, fibrinogen, andcombinations thereof.
 6. A packaged hemostatic textile comprising: awoven or knitted textile material that activates hemostatic systems inan animal or human when the textile material is in contact with a woundon the animal or human, wherein the woven or knitted textile material ispackaged in a sterilized package, wherein the textile comprises bamboorayon fibers.
 7. The packaged hemostatic textile of claim 6, wherein thetextile material comprises thrombin or a fraction containing thrombin.8. The packaged hemostatic textile of claim 6, wherein the textilematerial comprises a hemostatic agent selected from the group consistingof rehydrated lyophilized platelets, rehydrated lyophilized blood cells,fibrin, fibrinogen, and combinations thereof.
 9. The packaged hemostatictextile of claim 6, wherein the textile material comprises glass fibers.10. A hemostatic textile comprising: glass fibers cowoven or knittedwith bamboo rayon fibers, wherein when the hemostatic textile is incontact with a wound on an animal or human, hemostatic systems areactivated in the animal or human.
 11. The hemostatic textile of claim10, wherein the glass fibers comprise continuous glass fibers.
 12. Thehemostatic textile of claim 10, comprising thrombin or a fractioncontaining thrombin.
 13. The hemostatic textile of claim 10, comprisinga hemostatic agent selected from the group consisting of rehydratedlyophilized platelets, rehydrated lyophilized blood cells, fibrin,fibrinogen, and combinations thereof.
 14. The hemostatic textile ofclaim 10, wherein the glass fibers have a diameter of between 5nanometers and 15 microns.
 15. The hemostatic textile of claim 10,wherein the glass fibers comprise aluminoborosilicate glass.